Our studies have shown that age accounts for a significant portion of testicular variation in normal men, but much of the variation in human spermatogenesis remains to be explained. The long-term objective is to understand the variation in human spermatogenesis and its age-related reduction. The specific aims are to relate the variation in daily sperm production to: the architecture of tubules in terms of size and distribution of stages; stage-dependency of protein and RNA synthesis in Sertoli cells; DNA repair capacity of Sertoli cells or spermatogonia; biochemical composition of tubular boundary tissue; status of the blood testis barrier and amount of plasminogen activator in degenerative tubules; gene expression and chromosomal status of spermatocytes known to degenerate in aging men; in vitro production of androgens; and serum concentrations of FSH, LH, and testosterone. The experimental design for achieving these goals includes the use of autopsy specimens from men 20 to 94 years old and fresh specimens obtained from men aged 50 to 90 years undergoing elective orchidectomy for prostatic carcinoma. The unavailability of fresh specimens from young men necessitates the use of spermatogenically active testes from older men. The methods to be used include three-dimensional reconstruction of human seminiferous tubules; testicular stereology; testicular cell counts; autoradiography; radioimmunoassay; high performance liquid chromatography; chromosomal analysis; in vitro production of proteins, RNA, and androgens; and germ cell isolation by centrifugal elutriation followed by mRNA isolation and hybridization and in situ hybridization to evaluate the relationship between spermatocyte development or degeneration and gene expression during meioses. This project will lead to a better understanding of molecular mechanisms that influence human spermatogenesis and its age-related dysfunction.